Cells cycle through various stages of growth, starting with the M phase, where mitosis and cytoplasmic division (cytokinesis) occurs. The M phase is followed by the G1 phase, in which the cells resume a high rate of biosynthesis and growth. The S phase begins with DNA synthesis, and ends when the DNA content of the nucleus has doubled. The cell then enters G2 phase, which ends when mitosis starts, signaled by the appearance of condensed chromosomes. Terminally differentiated cells are arrested in the G1 phase, and no longer undergo cell division.
The hallmark of a malignant cell is uncontrolled proliferation. This phenotype is acquired through the accumulation of gene mutations, the majority of which promote passage through the cell cycle. Cancer cells ignore growth regulatory signals and remain committed to cell division. Classic oncogenes, such as ras, lead to inappropriate transition from G1 to S phase of the cell cycle, mimicking proliferative extracellular signals. Cell cycle checkpoint controls ensure faithful replication and segregation of the genome. The loss of cell cycle checkpoint control results in genomic instability, greatly accelerating the accumulation of mutations which drive malignant transformation. Thus, modulating cell cycle checkpoint pathways and other such pathways with therapeutic agents could exploit the differences between normal and tumor cells, both improving the selectivity of radio- and chemotherapy, and leading to novel cancer treatments. As another example, it would be useful to control entry into apoptosis.
On the other hand, it is also sometimes desirable to enhance proliferation of cells in a controlled manner. For example, proliferation of cells is useful in wound healing and where growth of tissue is desirable. Thus, identifying modulators which promote, enhance or deter the inhibition of proliferation is desirable.
Proteins of general interest that have been reported on include kinases. The Ste20 family of kinases can be divided into two structurally distinct subfamilies. The first subfamily contains a C-terminal catalytic domain and an N-terminal binding site for the small G proteins Rac1 and Cdc42 (Herskowitz, Cell, 80:187-197 (1995)). The yeast serine/threonine kinase Ste20 and its mammalian homologue, p21 Activated Kinase 1 (PAK1), belong to this subfamily. Ste20 initiates a mitogen-activated protein kinase (MAPK) cascade that includes Ste11 (MAPKKK), Ste7 (MAPKK), and FUS3/KSS1 (MAPK) in response to activation of the small G protein Cdc42, as well as signals from the hetero-trimeric G proteins coupled to pheromone receptors (Herskowitz, Cell, 80:187-197 (1995)). Similar to Ste20 , PAK1 has been reported to be a Cdc42 and Rac1 effector molecule and specifically regulates the c-Jun N-terminal kinase (JNK) pathway, one of the mammalian MAPK pathways (Bagrodia, et. al., J. Biol. Chem., 270:27995-27998 (1995); Kyriakis, et al., J. Biol. Chem., 271:24313-24316 (1996)). The JNK pathway is activated by a variety of stress inducing agents, including osmotic and heat shock, UV irradiation, protein inhibitors and pro-inflammatory cytokines such as tumor necrosis factor (TNF) (Ip, et al., Curr. Opin. Cell Biol., 10:205-219 (1998)). JNKs are activated through threonine and tyrosine phosphorylation by MEK4 and MEK7 (MAPKK), which are in turn phosphorylated and activated by MAPKKKs including MEK kinase 1 (MEKK1), and mixed lineage kinases MLK2 and MLK3 (Ip, et al., Curr. Opin. Cell Biol., 10:205-219 (1998)). In addition to the activation of the JNK pathway, PAK1 has also been reported to be a regulator of the actin cytoskeleton (Sells, et al., Curr. Biol., 7:202-210 (1997)).
The second subgroup of Ste20 family of kinases is represented by the family of germinal center kinases (GCK) (Kyriakis, J. Biol. Chem., 274:5259-5262 (1999)). In contrast to Ste20 and PAK1, GCK family members have an N-terminal kinase domain and a C-terminal regulatory region. Many GCK family members, including GCK, germinal center kinase related protein (GCKR), meatopoietic protein kinase (HPK) 1, GCK-like kinase (GLK), HPK/GCK-like kinase (HGK) and NCK interacting kinase (NIK), have also been reported to activate the JNK pathway when overexpressed in 293 cells (Pombo, et al., Nature, 377:750-754 (1995); Shi, et al., J. Biol. Chem., 272:32102-32107 (1997); Kiefer, et al., EMBO J. 15:7013-7025 (1996); Diener, et al., Proc. Natl. Acad. Sci. USA, 94:9687-9692 (1997); Yao, et al., J. Biol. Chem., 274:2118-2125 (1999); Su, et al., EMBO J., 16:1279-1290 (1997)). Among those, GCK and GCKR have been implicated in mediating TNF-induced JNK activation through TNF receptor associated factor 2 (Traf2) (Pombo, et al., Nature, 377:750-754 (1995); Diener, et al., Proc. Natl. Acad. Sci. USA, 94:9687-9692 (1997); Yuasa, et al., J. Biol. Chem., 273:22681-22692 (1998)). NCK interacting kinase (NIK) interacts with the SH2-SH3 domain containing adapter protein NCK and has been proposed to link protein tyrosine kinase signals to JNK activation (Su, et al., EMBO J., 16:1279-1290 (1997)).
A kinase related to TNIK has been reported on. MINK (misshapen/NIKs-related kinase) protein and nucleic acid have been previously described (Ippeita et. al., FEBS Letters, 469:19-23, 2000). MINK1 is a gck kinase family member which is upregulated during brain development (Ippeita et. al., FEBS Letters, 469:19-23, 2000).
One study reports on a GCK family kinase from Dictyostelium that can phosphorylate Severin in vitro. (Eichinger, et al., J. Biol. Chem., 273:12952-12959 (1998)). Severin is an F-actin fragmenting and capping enzyme that regulates Dictyostelium motility. TNIK, a mammalian GCK, has been shown to regulate the cytoskeleton, particularly to destabilize F-actin (Fu et al., JBC 274:30729-30737, 1999).
The Rho, rac and cdc42 small GTPases have been shown to regulate actin polymerization and the formation of multimolecular focal complexes (for example, see Nobes et al., Cell 81:53-62, 1995, and references therein; incorporated herein by reference). Further, PAK1 has been shown to regulate actin cytoskeleton organization, possibly through the phosphorylation and inhibition of the myosin light chain kinase Sanders et al., Science 283:2083-2085, 1999).
In addition, intracellular signaling mechanisms affecting cytoskeletal organization and underlying cell migration in response to extracellular cues have been studied in some detail (for review, see Maghazachi et al., Int. J. Biochem. Cell Biol. 32:931-943, 2000).
Several kinases and other intracellular signaling molecules have also been implicated in the control of apoptosis and cell survival in mammalian cells. For example, the JNK family of kinases has been implicated in both apoptosis and cell survival, the particular effect being dependent on the cellular context (for review, see Ip et al., Curr. Opin. Cell Biol. 10:205-219, 1998).
The role of GCKs in the immune system is of particular interest. Although GCKs are expressed widely, in B lymphocytic follicular tissue, GCK expression is largely restricted to the germinal center (Katz et al., JBC 269:16802-16809, 1994). In germinal centers, B lymphocytes undergo differentiation and selection, which is induced in part by ligands including members of the TNF family. These ligands activate GCKs which in turn activate other protein kinases that induce lymphocyte development (reviewed in Kyriakis, JBC 274:5259-5262, 1999).
The integrity of intracellular signal transduction pathways and their appropriate regulation is essential for B cell and T cell development and function. An understanding of these signaling pathways is therefore desirable to provide means for therapeutically modulating lymphocyte function in a variety of disorders characterized by hyper immune responses (e.g. auto-immune disorders) or hypo immune responses (e.g. immunodeficiency disorders). Such understanding is also desirable to provide for the modulation of normal but undesirable immune responses, for example following transplant immunosuppressive agents are desirable.
The modulation of signal transduction, proliferation, apoptosis, morphological change, and migration in mammalian cells is desirable, for example for the treatment of cancer, immune dysfunction, and for immunosuppression. Accordingly, compositions and methods for modulating these processes in mammalian cells are desirable.